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WO1997043410A1 - Gene de susceptibilite retrovirale et utilisation de ce gene - Google Patents

Gene de susceptibilite retrovirale et utilisation de ce gene Download PDF

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Publication number
WO1997043410A1
WO1997043410A1 PCT/GB1997/001311 GB9701311W WO9743410A1 WO 1997043410 A1 WO1997043410 A1 WO 1997043410A1 GB 9701311 W GB9701311 W GB 9701311W WO 9743410 A1 WO9743410 A1 WO 9743410A1
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Prior art keywords
leu
polypeptide
ser
seq
glu
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Jonathan Paul Stoye
Paul Roussel Le Tissier
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Medical Research Council
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the provision of genes which inhibit retroviruses in cells, specifically the Fvl gene, and uses thereof, including in assays for molecules useful in retroviral inhibition and in therapeutic protocols.
  • Retroviral infections in mice are controlled by a number of genes including the Friend virus susceptibility-1 ( Fvl ) gene, which acts to prevent retrovirus integration in newly infected cells.
  • Fvl is related to one of the many endogenous retroviruses present in the genomes of vertebrates and apparently represents an example of the opportunistic use of a gene originally derived from one family of mobile genetic element to combat another class of infectious element.
  • a substantial fraction of the mammalian genome consists of endogenous retrovirus-like elements (1) . Study of these proviruses provides compelling evidence that vertebrate evolution has taken place against a background of constant retrovirus infection (2) . It is therefore not surprising that a number of host genes controlling retrovirus replication have evolved (3) .
  • One such gene is Friend virus susceptibility-1, Fvl (4) which maps to mouse chromosome 4 (5) . It is a dominant resistance gene with the property of acting on infecting virus at a stage after entry into the target cell but prior to integration and formation of the provirus (6) .
  • the majority of inbred strains carry either the Fvl n or the Fvl b alleles.
  • Fvl n genotype are susceptible to murine leukaemia viruses (MLVs) called N-tropic and resistant to B-tropic MLVs (7, 8) .
  • MLVs murine leukaemia viruses
  • Fvl b mice show the reciprocal pattern.
  • NB-tropic viruses are not restricted by either allele.
  • restriction is not absolute, Fvl can prevent or delay spontaneous or experimentally induced viral tumours (9) .
  • Fvl restriction is manifested by an apparent 50-1000 fold reduction in viral titre (10) .
  • Genetic evidence implicates a direct interaction between the Fvl gene product and a component of the viral preintegration complex, the capsid protein, CA (11-14) .
  • the present invention thus provides a polypeptide in substantially isolated form comprising a amino acid sequence having at least about 60% sequence identity to the polypeptide of SEQ ID NO. 2 or SEQ ID NO. 4, or a fragment thereof. Preferred fragments are those having the ability to inhibit retroviral replication.
  • the polypeptide consists essentially of the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4.
  • the polypeptide may be in the form of a fusion protein comprising a polypeptide of the invention fused to a second polypeptide, such as a DNA binding domain, an activation domain, an antibody or binding fragment thereof, or a detectable label such as an epitope or an enzyme.
  • the invention further provides an isolated nucleic acid comprising a sequence of nucleotides encoding a polypeptide of the invention.
  • a nucleic acid includes a sequence of nucleotides comprises the open reading frame of SEQ ID NO. 1 or SEQ ID NO. 3.
  • the nucleic acid may be incorporated into an expression vector where it is under the control of a promoter.
  • Such a vector may be introduced into a host cell, the promoter of said vector being compatible with the host cell .
  • the host cell may be used for a variety of purposes, including for the preparation of polypeptides of the invention by a method comprising culturing said host cell under conditions in which said polypeptide is expressed, and recovering the polypeptide.
  • a particular application of the present invention resides in the provision of an assay for an agent capable of inhibiting the growth or replication of retroviruses which comprises bringing a polypeptide of the invention into contact with a retroviral protein and determining the degree of interaction between the two proteins.
  • the polypeptide of the invention may be obtainable by any suitable means, including: a) preparing a fragment of from 8 to 40 amino acids of the polypeptide of SEQ ID NO. 2 or SEQ ID NO. 4; or b) preparing a polypeptide having the sequence of the polypeptide of (a) except for from 1 to 4 amino acid substitutions.
  • nucleic acid includes both DNA and RNA.
  • the present invention provides a nucleic acid isolate comprising a sequence of nucleotides encoding the amino acid sequence encoded by the coding region of SEQ ID NO. 1 or SEQ ID NO. 3.
  • the amino acid sequences of the coding regions of SEQ ID NO. 1 and SEQ ID NO. 3 are shown as SEQ ID NO. 2 and SEQ ID NO. 4 respectively. Differences between the amino acid sequence encoded by SEQ ID NO.'s 1 and 3 are shown in Table 1 below.
  • the coding sequence may comprise or consist essentially of a sequence of nucleotides encoding the amino acid sequence encoded by the nucleotides from position 2167 to 3543 in SEQ ID NO. 1 or the equivalent sequence of SEQ ID NO. 3 (positions 35 to 1354) .
  • the nucleic acid comprises or consists essentially of nucleotides numbered 2167-3543 of SEQ ID NO. 1 or the equivalent sequence of SEQ ID NO. 3.
  • Nucleic acid according to the present invention may comprise or consist of nucleotides encoding one or more fragments of the amino acid sequence encoded by SEQ ID NO. 1 or SEQ ID NO. 3, e.g sufficient to inhibit retroviral replication. Restriction enzyme or nucleases may be used to digest the nucleic acid, followed by an appropriate assay
  • a preferred embodiment of the present invention provides a nucleic acid isolate with the minimal nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3 required for anti-retroviral activity.
  • a sequence which encodes an amino acid sequence that is a fragment, mutant, allele, derivative or variant, by way of addition, insertion, deletion or substitution of one or more amino acids of the sequence encoded by SEQ ID NO. 1 or SEQ ID NO. 3 may be employed, it being preferred that the fragment, mutant, allele, derivative or variant polypeptide retains the ability to inhibit retroviral replication.
  • the amino acid sequence may have at least about 60% homology or identity with one of the sequences encoded by SEQ ID NO. 1 or SEQ ID NO. 3, preferably at least about 65%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95% identity.
  • the nucleic acid sequence in accordance with an embodiment of the invention may hybridise with one or both of the shown sequences, or the complementary sequences (since DNA is generally double-stranded) .
  • Fragments of polypeptides of the invention include anything from single amino acid truncations of the polypeptides of SEQ ID NO. 2 and SEQ ID NO. 4 to fragments of 5 amino acids in size. Thus fragments may be from at least 20 to 400, e.g. at least 50, 75, 100 or 150 amino acids in size. Such fragments are useful in, amongst other things, the production of antibodies. Fragments capable of inhibiting retroviral replication are preferably from 6 to 100 amino acids, for example from 6 to 40 or 8 to 100 amino acids, more preferably from 8 to 40 amino acids.
  • a suitable assay means includes the two-hybrid assay system described below, and the ability of a polypeptide of the invention to interact with a polypeptide component of a retrovirus in such a system is a suitable indicator of activity of the polypeptide of the invention.
  • the polypeptide of the invention may also be tested by expressing the polypeptide in a host cell and infecting the host cell with a retrovirus. This is analogous to the experiments conducted in the accompanying examples .
  • the host cells will be mammalian host cells, preferably rodent, primate or human and the retrovirus may be any retrovirus capable of infecting the host cell, e.g murine leukaemia viruses, HIV-1 and -2, HTLV-I and -II, SIV-1 and -2, mammalian endogenous retroviruses such as porcine and baboon endogenous retroviruses, and Feline leukaemia virus.
  • the virus may be naturally occurring or be modified in a routine manner to incorporate a marker gene allowing for simple assay of viral titre, such as a puromycin resistance gene.
  • Viral titre may be measured in a number of other ways using techniques known in the art for the assay of retroviral titres. This includes assaying reverse transcriptase (RT) for example RT activity in the supernatant of cell cultures. Another measure is that of newly synthesised nucleic acid in infected cells. Viral antigen production may also be measured as an indicator of viral titre.
  • Polypeptides of the invention may be in the form of fusion proteins in which polypeptide is fused, at the N- or C- terminus, or both, to other sequences.
  • Fusion proteins further include fusions with DNA binding domains and/or activation domains for two-hybrid assay systems as described herein. Further examples of fusion proteins include fusions with antibodies or binding fragments thereof, or with enzymes allowing detection of the polypeptides of the invention in an immunoassay system.
  • Fusion proteins also include two separate polypeptides of the invention or fragments thereof joined together by intervening polypeptide sequences, optionally with additional N-terminal or C-terminal (or both) polypeptide sequences fused to the N- or C- terminus of such a fusion protein.
  • modifications may be made at the nucleic acid level that are not reflected at the encoded amino acid sequence level. Such modifications may be by insertion, addition, deletion and/or substitution of one or more nucleotides of the coding sequence nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 3.
  • Nucleic acid according to the present invention may be in the form of a recombinant vector, for example a plasmid, phage or cosmid vector.
  • the coding sequence may be under the control of appropriate regulatory elements, such as promoter elements, for expression in a host cell, for example a mammalian or yeast cell. In the case of genomic DNA, this may contain its own promoter or other regulatory elements . Otherwise, e.g. for cDNA, the coding sequence may be under any control elements appropriate for the chosen host cell .
  • appropriate regulatory elements such as promoter elements
  • genomic DNA this may contain its own promoter or other regulatory elements .
  • the coding sequence may be under any control elements appropriate for the chosen host cell .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • a further aspect of the present invention provides a host cell containing a nucleic acid according to the present invention, following prior introduction of the nucleic acid into the cell or an ancestor thereof.
  • a still further aspect provides a method comprising introducing such nucleic acid into a host cell.
  • the introduction may employ any available technique, including, for eukaryotic cells, calcium phosphate transfection, DEAE-Dextran transfection, electroporation, liposome-mediated transfection and transduction using retrovirus.
  • the introduction may be followed by causing or allowing expression of the encoded sequence, e.g. by cultu ⁇ ng host cells under conditions for expression of the gene. Such expression may confer on the cell resistance to retroviral infection.
  • Expression may be for the purposes of preparing a polypeptide of the invention in which case the polypeptide may be recovered from the host cell. This may be done by any suitable means. For example where the polypeptide is expressed and secreted from the cell (possibly due to the presence of a suitable signal sequence) , it may be recovered from the surrounding media by standard purification techniques such as chromatography, e.g. immunoaffinity chromatography, to a degree suitable for the intended purpose of the particular polypeptide. Where the polypeptide is expressed as a fusion protein to assist recovery, the fused portions may be removed by chemical or enzymatic means if desired.
  • chromatography e.g. immunoaffinity chromatography
  • nucleic acid e.g. a construct comprising a promoter and coding sequence
  • the genome e.g. chromosome
  • Integration may be promoted by inclusion m the construct of sequences which promote recombination with the genome, in accordance with standard techniques.
  • nucleic acid into a host cell may be performed in vi tro or in vivo, e.g. in a gene therapy protocol as discussed further below.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al . eds. , John Wiley & Sons, 1992, the disclosure of which is incorporated herein by reference.
  • Nucleic acid molecules, constructs and vectors according to the present invention may be provided isolated and/or purified (i.e. from their natural environment) , in substantially pure or homogeneous form, free or substantially free of nucleic acid or genes of the species of interest or origin other than the promoter sequence. Nucleic acid according to the present invention may be wholly or partially synthetic. The term “isolate” encompasses all these possibilities.
  • nucleic acid encoding polypeptides of the invention which are not identical to the polypeptide sequences of SEQ ID NO. 2 or SEQ ID NO. 4 is required, this may be prepared by manipulation of the nucleic acid of SEQ ID NO. 1 or SEQ ID NO. 3 using conventional technology such a site-directed mutagenesis, or the introduction of synthetically-produced sequences in between defined restriction enzyme sites, which have been cut to remove the naturally occurring Fvl sequence.
  • the polypeptides may be produced synthetically, for example by step wise chemical synthesis. In this event, it is feasible to introduce non-naturally occurring amino acids, including D-isomers of naturally occurring amino acids, which may be incorporated into polypeptides of the invention to improve their stability in a cellular environment.
  • Fvl acts in the murine leukaemia virus system in functions (i) or (ii) .
  • the discovery of the principle of F l's action may be used in design of assay systems, such as cell-free systems, mammalian cell systems and yeast two-hybrid systems, to screen for interaction of naturally occurring and synthetic gag-like peptides, such as Fvl and modified versions thereof, with components of retroviruses. Interference with any of such interactions would forms basis for an antiviral screen.
  • a major use of the nucleic acid is in screening for substances able to modulate action of the encoded polypeptide or for modified versions of the polypeptide that are able to inhibit replication of different retroviruses. It is well known that pharmaceutical research leading to the identification of a new drug generally involves the screening of very large numbers of candidate substances, both before and even after a lead compound has been found.
  • a further aspect of the present invention provides a screening system that comprises the provision of Fvl polypeptide (or other gag-like peptide) or a fragment, mutant, allele or derivative thereof, e.g.
  • a library of molecules may be screened, either in a "combinatorial" approach against a library of potentially interacting molecules or in a simpler approach against a fixed counterpart.
  • Different gag-like peptides or Fvl fragments, alleles, derivatives or variants may be screened against the same retrovirus or component thereof, or different retroviruses or components thereof may be screened against Fvl or the same fragment, derivative, allele or variant thereof or other gag-like peptide.
  • other substances may be screened for ability to modulate interaction between Fvl (etc.) and the retrovirus or component thereof .
  • Interaction between Fvl or other molecule and retrovirus may be assessed by inhibition of retroviral replication in an appropriate host cell.
  • various assay systems may be employed, but one particularly suitable system may be the yeast two-hybrid system.
  • yeast two-hybrid system (Evan et al. , (1985) J. Mol . Cell Biol . 5: 3610-3616; Fields and Song (1989) Nature 340: 245-246) utilises a yeast containing a GAL4 responsive promoter linked to beta-galactosidase gene and to a gene (His3) that allows the yeast to grow in the absence of the amino acid histidine and to grow in the presence of the toxic compound 3-aminotriazole.
  • Nucleic acid encoding test polypeptides are cloned into yeast vectors that express the proteins as fusions with the DNA binding domain of GAL4 or, e.g.
  • GAL4 activator fusions in the case of a library to be screened, GAL4 activator fusions.
  • a blue colour in yeast on indicator plates due to activation of beta-galactosidase, the yeast being able to grow in the absence of histidine, is indicative of interaction between the two components (e.g. Fvl and retroviral component) . This interaction is in turn indicative of potential anti-retroviral activity of the test polypeptide derived from or similar to Fvl, e.g. in the library.
  • DBD DNA binding domains
  • TAD transcriptional activation domains
  • the interaction of the domains may be measured by the use of other suitable reporter genes operably linked to a DNA binding sites compatible with the DBD.
  • the viral VP16 activation domain Fields and Jang, 1990
  • Knowledge of action of the Fvl gene product may be used to develop screens for interaction between retroviral proteins and host cell factors.
  • the Fvl protein, naturally-occurring related proteins or synthetic adaptations of these may be used to look for their ability to interact with and preferably restrict replication of retroviruses , e.g. other than the murine leukaemia viruses, including for example, any of the retroviruses mentioned above.
  • Assays developed from the provision of Fvl by the present invention may be used to investigate naturally occurring resistance to retroviral infection found amongst the human population. Once suitable reagents have been identified they may be tested in gene and pharmaceutical therapeutic programmes. Those skilled in the art are well aware of a multitude of possible reporter genes and assay techniques which may be used to determine gene activity. Any suitable reporter/assay may be used and it should be appreciated that no particular choice is essential to or a limitation of the present invention.
  • Molecules identified as being able to interact with a component of a retrovirus and/or inhibit retroviral replication individually represent further aspects of the present invention. If peptides or polypeptides, they may be produced by expression from encoding nucleic acid for subsequent use as desired, for instance in a manufacture of a medicament or in a therapeutic protocol as discussed further below. Novel variants and derivatives of the polypeptides encoded by SEQ ID NO. 1 and SEQ ID NO. 3 may be made by manipulation of the encoding nucleic acid, as discussed, and also represent individually further aspects of the present invention, especially if they have identifiable ability to interact with a component of a retrovirus and/or inhibit retroviral replication.
  • Such a process may include the step of :
  • Polypeptides identified in (iii) as active in interacting with a particular retrovirus may be used as a starting point for the further production of variants with improved interaction with the same retrovirus or the ability to interact with retroviruses of different species.
  • the variants of (iii) may be prepared by any of the processes described herein for the production of polypeptides and fragments thereof of the invention. This includes recombinant production or chemical synthesis.
  • the variants may be random mutants, obtained by for example random mutagenesis of nucleic acid encoding the active fragments identified in (ii) or by introducing random amino acids in one or more steps of the step wise synthesis of polypeptides.
  • Variants may also be designed so the particular residues of the fragments identified as active in step (ii) are either conserved or mutated, and such mutations may be entirely at random or be substitutions by conserved residues, e.g. to maintain a charged amino acid at a predetermined position. Mutations may also be designed to conserve features of secondary structure where such structure is predicted by computer algorithms, e.g by testing mutations against the same algorithm.
  • the substance may be manufactured (e.g. by a process comprising expression from encoding nucleic acid, in the case of a peptide or polypeptide) and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention extends in various aspects not only to a substance identified using the teaching disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for inhibiting retroviral replication, use of such a substance in manufacture of a composition for administration, e.g. for inhibiting retroviral replication, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • Administration will preferably be in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions according to the present invention may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • a mimetic or mimic or the substance may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • a further aspect of the invention provides antibodies against the novel polypeptides of the invention.
  • Such antibodies which may be monoclonal or polyclonal, may be prepared by standard immunological techniques .
  • an antibody may be natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies.
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A- 0120694 and EP-A-0125023.
  • Nucleic acid according to the present invention may be used a gene therapy protocol that aims for delivery of the nucleic acid to target cells.
  • Nucleic acid may be introduced into cells in vi tro, the cells then being transplanted or grafted into the body, perhaps back into the source body (which has the advantages of autologous transplantation) , or the nucleic acid may be administered to an individual in a form or with a suitable carrier for delivery to target cells.
  • nucleic acid encoding a peptide or polypeptide that is able to interact with a component of a retrovirus may be introduced into the genome of immune system stem cells removed from a patient infected with a virus, such as HIV.
  • the encoded gene product may be an Fvl polypeptide or fragment or derivative etc. thereof, or it may be a polypeptide or peptide identified using a screen (discussed above) as having ability to interact with a component of a retrovirus (e.g. it may be a gag-like polypeptide or fragment thereof) .
  • immune system cell descendants of the transgenic stem cells may be resistant to retroviral infection.
  • peptides and polypeptides may be delivered on liposomes, fused to antibodies or binding fragments thereof specific for the target cells (e.g. cells infected with retrovirus) .
  • Nucleic acid may be delivered naked or using a viral vector (e.g. adenoviral or retroviral) or other carrier, such as a liposome. Intravenous injection of constructs in cationic liposomes has resulted in widespread transfection of most tissues. Lack of immunogenicity of liposomes allows for repeated administration.
  • Part A Isolation of a YAC carrying the Fyl crene
  • chromosome 4 Detailed genetic analyses of the distal region of chromosome 4 have identified two markers, the endogenous provirus, Xmv9 , and the gene for atrial natriuretic factor, Nppa , which are likely to lie within 1.2 Mb of Fvl (15, 16) . We therefore set out to isolate YACs corresponding to these markers and build a contig spanning the region in which Fvl must lie. Since Fvl is a dominant gene, introduction of YACs containing the gene into cell lines should result in alterations in susceptibility to MLV infection, thereby allowing a direct assay for the presence of Fvl .
  • YAC libraries Three YAC libraries (17-19) were screened by PCR for X v9 and Nppa . All three libraries were made with DNA from C57BL6 mice and would therefore contain the Fvl b allele. A number of YACs, ranging in size from 120kb to 900kb, were isolated. Many of the larger YACs were unstable. None contained both Xmv9 and Nppa . We chose to work with the largest stable YACs for the two markers; Xmv9 Jl (350kb) and B2 (250kb) as well as Nppa G7 (210kb) and Dll (190kb) . To allow selection of these YACs, the Neo R gene was inserted into the right arm of the YACs by homologous recombination with the plasmid pRVl (20) .
  • the next task was to identify a suitable cell line as recipient for YAC DNA following spheroplast fusion.
  • the ideal recipient would show not only a high frequency of G418 resistant colonies but also uptake of DNA corresponding to the whole YAC.
  • they should be of appropriate phenotype to allow detection of the introduced Fvl gene from YACs of Fvl b origin (i.e. they should be Fvl n or not express Fvl ) .
  • Modified YACs were fused to three such cell lines, NIH-3T3, 3T3FL and L cells, and the frequency of Neo R colonies determined. Colonies were analysed by PCR for the presence of Nppa as well as markers in the left and right YAC arms.
  • L cells appeared the most suitable for our purposes. They are derived from C3H mice and should therefore be Fvl n (21) ; they yielded the highest frequency of Neo R colonies and the greatest percentage of potentially intact YACs as judged by the presence of Nppa and the left YAC arm (the selectable marker is present in the right arm) .
  • L cells did however introduce a further complication as they spontaneously produce high levels of endogenous ecotropic MLV (22) .
  • virus production does not prevent cells exhibiting Fvl activity (23) , it makes them insensitive to infection by exogenous ecotropic virus since the cellular receptor for virus will be blocked by envelope protein from the endogenous virus - a phenomenon known as interference (24) . It also precludes any assays for Fvl activity based on reverse transcriptase.
  • YAC Xmv9 Jl was transferred to a RAD52 background by mating with a non-YAC containing RAD52 strain followed by sporulation and tetrad dissection. YACs were then modified to carry the Neo R gene by homologous recombination with linearised pRVl (20) . Yeast spheroplasts were fused with mouse cells essentially as previously described (36) and colonies of neomycin resistant cells isolated after 2 weeks growth in 800mg/ml G418.
  • Virus stocks were recovered by transfecting M. dunni cells with cloned DNAs using Lipofectin followed by twice-weekly passaging until maximum supernatant reverse transcriptase (37) levels were seen.
  • Polytropic N- and B- tropic MLVs were constructed in vitro by replacing a 2.9kb Pstl (in the LTR) - Hindlll (in pol ) fragment from p247-W (38) with the analogous Pstl-Hindlll fragments from the ecotropic N- (pWN41) and B-tropic (pWB5) MLV clones (39) .
  • Virus stocks conferring resistance to puromycin were prepared by co-transfection of pBABEpuro (25) with different helper viruses, followed by selection for 14 days in puromycin (5 ⁇ g/ml) , infection of fresh M. dunni cells and selection of reverse transcriptase positive, puromycin resistant colonies. Virus stocks were titrated on 3T3FL or M. dunni cells. Virus infections used 15 ⁇ g/ml polybrene.
  • a cosmid library was prepared from YAC Nppa Dll. Twenty randomly selected cosmids were introduced into NIH-3T3 cells and several cell clones from each transfection tested for Fvl activity. Three cosmids, D11C5, D11C11 and D11C44 were identified which carried activity. Restriction mapping showed that they shared a substantial overlap. Non-overlapping cosmids lacked Fvl activity. Unfortunately not all clones from a cosmid containing the gene gave a positive result (presumably due to different integration events) making it unwise to seek to delimit the boundaries of the active gene by identifying negative cosmids.
  • DllC5dN contained approximately 18.5kb of D11C5. It was found to transfer Fvl activity.
  • DllSpE was only 6.5kb in length. It too could confer the ability to restrict N tropic MLV on recipient cells, indicating that the complete Fvl gene must lie within this 6.5kb Spel-EcoRI fragment of DNA.
  • a cosmid library from the C57BL6/J YAC Dll was prepared. Blocks of total yeast DNA containing YAC Dll were treated with increasing concentrations of Mbol and DNA from a portion of each block sized by pulsed field electrophoresis. DNA was extracted from the remaining portion of blocks which contained DNA of an appropriate size and cloned into SuperCosl (Stratagene) . The cosmid library was screened using random primed total mouse DNA. Overlapping cosmids were identified by side by side comparisons of Eco RI and Bglll digested cosmid DNAs.
  • DllC5dN was obtained following partial Notl digestion of a derivative of the starting cosmid into which a Notl site had been introduced via transposon mutagenesis (40) .
  • the second, DllSpEb represents an 6.5 kb Spel to EcoRI fragment from D11C5 cloned into pBK-CMV (Stratagene) .
  • the Fvl open reading frame was PCR amplified from DllSpEb using Pfu polymerase in combination with primers spanning the 5' (5' -GACGCGCTAGCCGAGTTCTAGGGAAA-3' ; SEQ ID NO.
  • Fvl containing clones were introduced into M. dunni or NIH-3T3 cells, individual G-418 resistant cell clones selected and tested for Fvl by infection with N, B and NB- ropic MLVs followed three days later by reverse transcriptase assays.
  • sequence of a 6.48kb Spel-EcoRI fragment was determined by the dideoxy method with Sequenase 2.0 from M13 subclones or single stranded phage DNA rescued from fl-origin containing plasmids using helper phage M13 K07.
  • Part C Comparison of n and b alleles of Fyl .
  • a lambda library prepared with AKR DNA was screened using a single copy probe located just upstream of the open reading frame. This probe reacts with a 12 kb EcoRI fragment in C57BL/6 DNA but we recovered a 9 kb fragment in the cloning experiment. Restriction analysis and sequencing studies showed two gross changes in the DNA structure between the corresponding fragments isolated from AKR and C57BL/6. The first was the insertion of an Intracisternal A type Particle (IAP) element about 1.8 kb downstream from the open reading frame within a pair of B2 repeats . This would not be expected to affect the protein product of the putative Fvl gene. The second change is a 1.3 kb deletion of DNA in AKR compared to C57BL/6.
  • IAP Intracisternal A type Particle
  • DBA/2 has the same predicted protein product as AKR but differed in non-coding sequences in that it lacked the IAP element found in AKR and C3H. We assume that the presence or absence of this element is responsible for the subtle difference in phenotype between DBA/2 and other Fvl n strains, perhaps acting to influence mRNA stability.
  • the AKR/J Fvl clone was isolated from an Eco RI library prepared in the lambda vector DASH II (Stratagene) .
  • the library was screened using a 190bp fragment (synthesised by PCR using the primers 5' -TTAGCCTGGAGCTCCTGAAG-3 ' ; SEQ ID NO. 9) and 5 ' -GAAGCGGAAGAAGTCTCTTG-3 ' ; SEQ ID NO. 10) which maps to the extreme right hand end of D11C5.
  • a region extending through the Fvl open reading frame into the IAP LTR was sequenced (EMBL Accession Number X97720 and SEQ ID NO. 3) .
  • the DBA/2 allele was synthesised by long PCR using primers 5' -GAAGCGGAAGAAGTCTCTTG-3' ; SEQ ID NO. 11, plus 5' -CATTTGCTCATCTGTGCCAG-3' ; SEQ ID NO. 12, and has also been sequenced.
  • the BALB/c and C3H alleles were synthesised by long PCR and examined by restriction mapping.
  • the activity of the AKR Fvl open reading frame was assayed in the same manner as for Fvl b using a subclone in pCI-neo.
  • Part D Transcription of the Fyl crene .
  • HERV-L 29 Homology was detected with two expressed sequence tags (Accession Numbers X71645 and R99971) as well as a genomic clone (X89211) representing a member of the human endogenous retrovirus family, HERV-L 29.
  • Fvl and the endogenous human retrovirus, HERV-L are 60% identical over a stretch of 1300bp. Although this region of the sequenced member of the HERV-L family does not encode an open reading frame, presumably reflecting mutagenic forces acting over time on an endogenous retrovirus, and shows no discernible sequence homology to other retroviruses, it seems likely, based on its position between an LTR and a pol gene, that it corresponds to the gag gene of the HERV-L family. The homology between Fvl and HERV-L would therefore suggest that the Fvl gene product is gag-like and shares some structural similarity with its target, the CA protein of MLV.
  • gag may be important for virus restriction, for example to permit binding of Fvl to CA, though there are hundreds of retroviral elements more closely related to MLV than Fvl and none of these restrict replication, which suggests there are additional features unique to Fvl . More detailed studies of the mechanism of Fvl restriction are now enabled by possession of the cloned gene.
  • Fvl appears to be derived in some unknown fashion from a retrovirus.
  • the overall structure of the Fvl locus appears similar in Mus dunni (not shown) and Mus musculus (C57BL/6) mice implying that the original integration event preceded the divergence of the progenitors of the two species and consequently that the Fvl gene is widely distributed among Mus species.
  • Endogenous MLVs are much less widely distributed (30) ; they appear to have colonised the Mus germ line more recently.
  • Fvl activity is found only in strains containing endogenous MLVs (31) suggesting that the anti-viral activity may have evolved in response to the presence of MLVs.
  • the apparently still more recent generation of the Fvl n allele suggests that this may be an on-going process. It is now possible to test for continuation of this process in vi tro to generate an Fvl gene with specificity for other retroviruses.
  • TTTTCTCTGC CACAAGTCTT CTCCTGGTCA ATTCATTTCT TTGCTTTTCT TTCTTTCTTT 660 CTTCCTTTCT TTAATTTGCA TGTATATCCC AAGGTCACAC AGCTACTAAA TGTCAAAAGC 720
  • CTCCCTCCAC AAGGTTGATT TAAACTATGT CCAGGGGAGA GCATTCTCAG ATGGAGCTGC 900
  • TTC AAA CCT GAA CTT GCC GAG GAC TCT CCG GAT AAT GAC TCT CCG GAT 2256 Phe Lys Pro Glu Leu Ala Glu Asp Ser Pro Asp Asn Asp Ser Pro Asp 15 20 25 30
  • GCA AAG GAA CAT GCT AGG AAA TGG ATT TTA AGG GTG TGG GAT AAT GGT 3024 Ala Lys Glu His Ala Arg Lys Trp He Leu Arg Val Trp Asp Asn Gly 275 280 285
  • AGA TGG CCT ACT ACA AGA GAG CTG CAG TCG CCT GAC ACC CTG GAG TGG 3216 Arg Trp Pro Thr Thr Arg Glu Leu Gin Ser Pro Asp Thr Leu Glu Trp 335 340 345 350
  • AGT TTA AAG CCA ACA GCA GCT GGC TTG ACT TCT GTA GGC TCT GTG GGG 3504 Ser Leu Lys Pro Thr Ala Ala Gly Leu Thr Ser Val Gly Ser Val Gly 435 440 445
  • CTTCAAAAAC CTCGGAGGCC ACAGAAAATG GCATTTAAAG GTGTTTTTAT TAATTTAAGG 4273
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Mus musculus
  • AAA CAG ACT GAA GCC CTC ATT GTG AGG TTA GCT GAT GTG CAA TCC CAG 484 Lys Gin Thr Glu Ala Leu He Val Arg Leu Ala Asp Val Gin Ser Gin 135 140 145 150
  • TGT CCA CAG TGG AGG GGC CCG GAA GAT GTA CCC ATC ACG AGA GCT ATG 1204 Cys Pro Gin Trp Arg Gly Pro Glu Asp Val Pro He Thr Arg Ala Met 375 380 385 390
  • TTCGCCGTTA CAAGATGGCG CTGACAGCTG TGTTCTAAGT GGTAAACAAA TAATCTGCGC 1824

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Abstract

L'invention porte sur des polypeptides isolés (SEQ ID NOs. 2 et 4) et sur les allèles et fragments de ces polypeptides qui sont des produits du gène de susceptibilité rétrovirale, les séquences SEQ ID NOs. 2 et 4 étant des protéines Fv1 (virus de Friend1) de souris. L'invention concerne les acides nucléiques codant ces polypeptides et l'utilisation de ces polypeptides et acides nucléiques, notamment dans les dosages d'inhibiteurs des rétrovirus.
PCT/GB1997/001311 1996-05-14 1997-05-14 Gene de susceptibilite retrovirale et utilisation de ce gene Ceased WO1997043410A1 (fr)

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Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
A. CORDONNIER ET AL.: "Isolation of novel human endogenous retrovirus-like elements with foamy virus-related pol sequence", J. VIROLOGY, vol. 69, no. 9, September 1995 (1995-09-01), AM.SOC.MICROBIOL.,WASHINGTON,US, pages 5890 - 5897, XP002037948 *
FRANKEL, WAYNE N. ET AL: "Genetic analysis of endogenous xenotropic murine leukemia viruses: association with two common mouse mutations and the viral restriction locus Fv-1", J. VIROL. (1989), 63(4), 1763-74 CODEN: JOVIAM;ISSN: 0022-538X, 1989, XP002037947 *
J.M. COFFIN: "Retrovirus restriction revealed", NATURE, vol. 382, 29 August 1996 (1996-08-29), MACMILLAN JOURNALS LTD., LONDON,UK, pages 762 - 763, XP002037952 *
L. BÉNIT ET AL.: "Cloning of a new murine endogenous retrovirus MuERV-L, with strong similarity to the human HERV-L element and with gag coding sequence closely related to the Fv1 restriction gene", J. VIROLOGY, vol. 71, no. 7, July 1997 (1997-07-01), AM.SOC.MICROBIOL.,WASHINGTON,US, pages 5652 - 5657, XP002037954 *
S. BEST ET AL.: "Positional cloning of the mouse retrovirus restriction gene Fv1", NATURE, vol. 382, 29 August 1996 (1996-08-29), MACMILLAN JOURNALS LTD., LONDON,UK, pages 826 - 829, XP002037951 *
S.P. GOFF: "Operating under GAG order: a block against incoming virus by the Fv1 gene", CELL, vol. 86, 6 September 1996 (1996-09-06), CELL PRESS,CAMBRIDGE,MA,US;, pages 691 - 693, XP002037953 *
STOYE J P ET AL: "Genetic map of the region surrounding the retrovirus restriction locus, Fv1, on mouse chromosome 4.", MAMMALIAN GENOME 6 (1). 1995. 31-36. ISSN: 0938-8990, XP002037946 *
T. PINCUS ET AL.: "A major genetic locus affecting resistance to infection with murine leukemia viruses II", J. EXP. MED., vol. 133, no. 6, 1 June 1971 (1971-06-01), ROCKEFELLER UNIV. PRESS, US, pages 1234 - 1241, XP002037949 *
W.P. ROWE ET AL.: "A major genetic locus affecting resistance to infection with murine leukemia viruses III", J. EXP. MED., vol. 137, no. 3, 1 March 1973 (1973-03-01), ROCKEFELLER UNIV. PRESS, US, pages 850 - 853, XP002037950 *

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